Preparation and characterization of methylene blue nanoparticles for Alzheimer&#39;s disease and other tauopathies

ABSTRACT

A delivery system of coated and uncoated nanoparticles (NP) for delivery of methylene blue (MB). The delivery system was developed using PLGA-based polymer that was repeatedly shown to be biocompatible and biodegradable. The parameters of synthesized NPs were within the suitable range for BBB permeation—specifically, the NPs were monodispersed, with slight negative charge, and with the size within 100-150 nm range suitable for intravenous delivery and delivery to the brain. The coating on the nanoparticle did not have a significant impact on the nanoparticle size and zeta potential. Based on the immunoblotting experiments using AD cellular model, the GSH coated NPs were better in reducing tau levels compared to MB solution. In vitro BBB Transwell permeation study showed eight fold higher MB-NP permeation compared to the MB solution over 24 hours.

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention relates, generally, to treatment and prevention oftauopathies. More specifically, it relates to nanoparticles withimproved brain delivery for treatment of Alzheimer's disease and otherrelated disorders.

2. Brief Description of the Prior Art

Alzheimer's disease (AD) is known as the most common form of dementiaand with the increase in ageing population worldwide, the number ofpersons with dementia is also expected to increase. AD is associatedwith specific clinical and pathological features such as cognitiveimpairment and neurophsychiatric disturbances [1, 2]. Histologicalfeatures of AD include extraneuronal β-amyloid plaques and intraneuronalneurofibrillary tangles [3, 4]. More specifically, neurofibrillarytangles consist of bundles of paired helical filaments comprising ofabnormal aggregation of tau protein [3]. Tau is a neuronalmicrotubule-associated protein responsible for stabilization of axonalmicrotubules. Among other functions of tau protein are its role insignal transduction, plasma membrane and actin cytoskeletoninteractions, neurite outgrowth, anchoring of enzymes, and regulation ofvesicle transport [3]. However, at high concentration tau, oligomersexhibit toxicity and result in neurodegeneration [5].

Phenothiazines, such as methylene blue (MB), have been of interest dueto its ability to inhibit tau filament formation and reduce the effectsof oxidative stress; hence, MB is considered as a potential treatmentfor AD and other tauopathies [6-9]. MB is especially practical aspossible therapeutic drug because of its well-characterized action ofcGMP pathway inhibition and has been shown to improve oxygen consumptionin the brain, repair mitochondrial function, and improve cellularmetabolism [10-12]. Moreover, MB effects the three neurotransmittersystems important in AD, namely the following: cholinergic, resulting inimprovement of memory [13]; serotonergic, resulting in antidepressantactivity and increase of 5-hydroxyindole acetic acid (5-HIAA) [14]; andglutamatergic, resulting in enhanced memory retention and excitability[15, 16].

One of the challenges in targeting the brain in pharmaceutical therapyis the selectivity of the blood-brain barrier (BBB), which restrictsmost chemical agents from crossing into the brain tissue. MB is a highlyhydrophilic molecule that limits its ability to cross the hydrophobicBBB, although limited and variable penetration has been demonstrated[17, 18]. Pharmacokinetic study on MB by Peter et al. suggestsdistribution of intravenously or orally-administered MB not only in thebrain but also in various organs such as heart, lungs, liver, andkidneys, decreasing its bioavailability in the brain [19]. PLGAmicrospheres and nanoparticles (NP) have recently become of increasedinterest as efficient delivery vehicles for various drugs [20-22].Further, successful outcomes have been reported in brain-targeteddelivery using NPs with various coatings, such as TWEEN 80, thiamine,and conjugated with transferrin receptor specific antibody [24-30].

Targeting of the NPs to the brain by using reduced glutathione (GSH)coating is justified as the brain is particularly abundant in GSHtransporters and GSH, an intracellular antioxidant that protects thisorgan from reactive oxygen species (ROS) generated by high oxygenconsumption [23, 31-39]. Higher brain uptake of paclitaxel has beenshown using the glutathione-coated nanoparticles in the mouse model[40]. Furthermore, localization of GSH in the brain has beendemonstrated by numerous studies utilizing 99mTcmeso-hexamethylpropyleneamineoxime (HMPAO) for labeling brain tissues.Thus, glutathione-coated particles as a drug delivery vehicle made ofpoly(lactic-co-glycolide) (PLGA)-b-PEG are postulated to be effectivecarrier for various drugs because of low toxicity, controlled drugrelease, and reduced uptake by reticulo-endothelial system (RES) in vivoas compared to unmodified PLGA [23][30].

However, distribution of methylene blue (MB) into the brain is stilllimited due to its high hydrophilicity. Accordingly, what is needed is anovel nanoparticle formulation of MB to improve its delivery to thebrain, thus serving as an effective treatment option for Alzheimer'sdisease and other related disorders. However, in view of the artconsidered as a whole at the time the present invention was made, it wasnot obvious to those of ordinary skill in the field of this inventionhow the shortcomings of the prior art could be overcome.

While certain aspects of conventional technologies have been discussedto facilitate disclosure of the invention, Applicants in no way disclaimthese technical aspects, and it is contemplated that the claimedinvention may encompass one or more of the conventional technicalaspects discussed herein.

The present invention may address one or more of the problems anddeficiencies of the prior art discussed above. However, it iscontemplated that the invention may prove useful in addressing otherproblems and deficiencies in a number of technical areas. Therefore, theclaimed invention should not necessarily be construed as limited toaddressing any of the particular problems or deficiencies discussedherein.

In this specification, where a document, act or item of knowledge isreferred to or discussed, this reference or discussion is not anadmission that the document, act or item of knowledge or any combinationthereof was at the priority date, publicly available, known to thepublic, part of common general knowledge, or otherwise constitutes priorart under the applicable statutory provisions; or is known to berelevant to an attempt to solve any problem with which thisspecification is concerned.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIG. 1 depicts an exemplary structure of methylene blue loaded in ananoparticle, according to an embodiment of the current invention. Thenanoprecipitation method discussed herein allows for the polymer to forma spherical nanoparticle with methylene blue trapped inside. The PEGgroups and carboxyl groups on the outside provide the specificproperties. The GSH coating remains on the outside of the nanoparticleand allows for interactions with receptors. COOH refers to a carboxylgroup; PEG refers to polyethylene glycol; and PLGA refers topoly(lactic-co-glycolic) acid.

FIG. 2 depicts morphology of the nanoparticles by electron microscopy.In images 1 a, 1 b, and 1 c, the NPs demonstrate consistent roundedmorphology throughout the sample. The polymer and PEG groups are clearlyvisible in forming of the outer edge of the nanoparticle. In images 2 a,2 b, and 2 c, the MB-NPs also demonstrate consistent round shape andsize. The loaded API is visible inside of the nanoparticle. In images 3a, 3 b, and 3 c, 2% GSH-coated MB-NPs retain the rounded appearance andremain consistent in the average size of the nanoparticle. GSH coatingis obvious in forming the sharp edge outer edge of the nanoparticle withevidenced increase in the average nanoparticle size.

FIG. 3 is a graphical illustration showing in vitro release profile ofcoated and uncoated MB-NPs (n=6). Cumulative release of the methyleneblue from the physical mixture of methylene blue and the polymer, fromthe nanoparticles, and from the coated nanoparticles is represented. Therate of release from the nanoparticle is faster within first 20 hoursand then slower as compared to the mixture. GSH coated nanoparticlesdemonstrated the slowest sustained release. The release profile confirmsthe presence of GSH coating. All values shown as a mean n=6 with ±SE.The acronyms can be seen as follows: nanoparticles (NPs), Methylene blue(MB), glutathione reduced (GSH), polydispersity index (PDI).

FIGS. 4A-4C depict nano-formulation of MB that reduce Tau. Cells wereharvested and samples were analyzed for tau levels by immunoblotting. InFIG. 4A, endogenous Tau-expressing human neuroblastoma SHSY-5Y cellswere treated with blank nanoparticles (np-Ctrl), MB-NPs (np-MB),GSM-coated NPs (G-np-MB), MB (in water, MB-sol), or vehicle (water,Ctrl) for 24 hours. In FIG. 4B, a similar experiment as in FIG. 4A wasperformed in HeLa cells stably transfected with human Tau(over-expressed tau). FIG. 4C is a quantitation plot of the immunoblotsafter GAPDH normalization. Statistical analyses across cell modelsdemonstrated that MB in solution and MB associated with nano-particleseach reduce tau levels slightly higher at 10 μM compared to 5 μM.

FIG. 5 depicts a setup of Transwell permeable support, suitable for a12-well plate. Both sides of the insert were coated with rat tailcollagen I (BD BIOSCIENCE) before seeding RBE4 and C6 cells to theirrespective sides in order to establish the co-culture.

FIG. 6 is a graphical illustration showing permeation of 10 μM of 2%glutathione-coated nanoparticles. 100 μL of the media was extracted frombelow the permeable support at approximately 0, 3, 6, and 24 hours;setup was replenished with fresh media. The results were quantified byUV spectroscopy at a wavelength of 600 nm. Quantification of the resultsand standard deviation represents an average of quantified triplicates(n=3).

FIG. 7 is a graphical illustration comparing permeation of 10 μM of freeMB drug solution versus 2% glutathione-coated NP. 100 μL of the mediawas extracted from below the permeable support at approximately 0, 3, 6,and 24 hours; setup was replenished with fresh media. The results werequantified by UV spectroscopy at a wavelength of 600 nm. Quantificationof the results and standard deviation represents average of quantifiedtriplicates (n=3). Fluorescence data regarding free drug solutionyielded by spectroscopy was reduced by 75% to mimic approximately 25%entrapment efficiency of the MB drug by NPs as indicated herein. Datafor the 2% glutathione-coated nanoparticles was statisticallysignificantly higher than data for the free MB drug solution (p<0.05).

BRIEF SUMMARY OF THE INVENTION

The long-standing but heretofore unfulfilled need for a more effectivedelivery system of methylene blue is now met by a new, useful, andnonobvious invention.

In an embodiment, the current invention is a composition comprisingmethylene blue admixed with a polymeric-based layer of nanoparticles,wherein the layer of nanoparticles substantially encloses the methyleneblue. The layer of nanoparticles includes an outer layer with an outersurface covalently modified by a spacer linked to a hydrophilic group,such that the spacer linked to the hydrophilic group extends outwardlyfrom the outer surface of the outer layer of the layer of nanoparticlesto facilitate penetration of the composition to a targeted region (e.g.,brain) of a subject.

The layer of nanoparticles modified by the spacer linked to thehydrophilic group may be a PLGA-PEG-COOH structure.

The spacer may contain a hydrocarbon chain having a multiple bond,wherein the hydrocarbon chain contains an R group selected from thegroup consisting of a hydrogen, alkyl, alkenyl, alkynyl, alkoxy,hydroxylalkyl, hydroxyl, and haloalkyl.

The spacer may be selected from the group consisting of modifiedpolyethylene glycols, propylene glycols, amino acids, peptides,chelators and polaxamers.

The hydrophilic group may be a carboxyl group.

The polymer-based layer of nanoparticles may include PLGA.

The outer surface of the layer of nanoparticles may be further modifiedby a hydrophobic, protective coating that facilitates distribution ofthe methylene blue in the targeted region of the subject.

The coating may be formed of glutathione, which permits controlled andsustained release of the methylene blue into the targeted region of thesubject.

In a separate embodiment, the current invention is composition foreffectively permeating or penetrating a blood-brain barrier of asubject. The composition comprises a pharmaceutical agent admixed with apolymeric-based layer of nanoparticles, wherein the layer ofnanoparticles substantially encloses the pharmaceutical agent. The layerof nanoparticles includes an outer layer with an outer surfacecovalently modified by a spacer linked to a hydrophilic group, such thatthe spacer linked to the hydrophilic group extends outwardly from theouter surface of the outer layer of the layer of nanoparticles.

The outer surface of the layer of nanoparticles is further modified by ahydrophobic, protective glutathione coating that stabilizes thecomposition during permeation or penetration of the blood-brain barrier.The composition further has a (slight) negative charge.

The pharmaceutical agent may be methylene blue.

The layer of nanoparticles modified by the spacer linked to thehydrophilic group may be a PLGA-PEG-COOH structure.

The spacer may contain a hydrocarbon chain having a multiple bond,wherein the hydrocarbon chain contains an R group selected from thegroup consisting of a hydrogen, alkyl, alkenyl, alkynyl, alkoxy,hydroxylalkyl, hydroxyl, and haloalkyl.

The spacer may be selected from the group consisting of modifiedpolyethylene glycols, propylene glycols, amino acids, peptides,chelators and polaxamers.

The hydrophilic group may be a carboxyl group.

The polymer-based layer of nanoparticles may include PLGA.

The composition may have a size or diameter of less than about 150 nm.

In a separate embodiment, the current invention is a composition forreduction of tau levels (e.g., treatment of Alzheimer's disease andrelated tauopathies) in a brain of a subject. The composition comprisesmethylene blue admixed with a polymeric-based layer of nanoparticles,wherein the layer of nanoparticles substantially encloses the methyleneblue. The layer of nanoparticles includes an outer layer with an outersurface covalently modified by a spacer linked to a hydrophilic group,such that the spacer linked to the hydrophilic group extends outwardlyfrom the outer surface of the outer layer of the layer of nanoparticles.The outer surface of the layer of nanoparticles is further modified by ahydrophobic, protective glutathione coating that stabilizes thecomposition during permeation or penetration of the blood-brain barrier.The glutathione coating also permits controlled or sustained release ofthe methylene blue in a brain of the subject.

The layer of nanoparticles modified by the spacer linked to thehydrophilic group may be a PLGA-PEG-COOH structure.

The spacer may contain a hydrocarbon chain having a multiple bond,wherein the hydrocarbon chain contains an R group selected from thegroup consisting of a hydrogen, alkyl, alkenyl, alkynyl, alkoxy,hydroxylalkyl, hydroxyl, and haloalkyl.

The spacer may be selected from the group consisting of modifiedpolyethylene glycols, propylene glycols, amino acids, peptides,chelators and polaxamers.

The hydrophilic group may be a carboxyl group.

The polymer-based layer of nanoparticles may include PLGA.

The composition may have a size or diameter of less than about 150 nm.

These and other important objects, advantages, and features of theinvention will become clear as this disclosure proceeds.

The invention accordingly comprises the features of construction,combination of elements, and arrangement of parts that will beexemplified in the disclosure set forth hereinafter and the scope of theinvention will be indicated in the claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the following detailed description of the preferred embodiments,reference is made to the accompanying drawings, which form a partthereof, and within which are shown by way of illustration specificembodiments by which the invention may be practiced. It is to beunderstood that other embodiments may be utilized and structural changesmay be made without departing from the scope of the invention.

Methylene blue (MB) has been shown to slow down the progression of theAlzheimer's disease (AD) and other tauopathies; however distribution ofMB into the brain is limited due its high hydrophilicity. As a result ofthe challenges in MB delivery to the brain, an object of the currentinvention is to improve BBB penetration and MB cellular uptake in thebrain and to decrease the systemic MB side effects. In an embodiment, ahydrophobic glutathione coated PLGA nanoparticles was developed toimprove bioavailability of MB in the brain. Glutathione coatedpoly-(lactide-co-glycolide) (PLGA-b-PEG) nanoparticles (NPs) wereprepared and tested in two different cell culture models of ADexpressing microtubule associated protein tau (tau). The NPs showed aparticle size averaging 136.5±4.4 nm, which is suitable for the bloodbrain barrier (BBB) permeation. The in vitro release profile of the NPsexhibited no initial burst release and showed sustained drug release forup to 144 hours.

In another embodiment, a delivery system for MB was developed forpurpose of reducing tau levels in the human brain, in order to treat orprevent Alzheimer's disease and other tauopathies. Unexpectedly,treatment with newly formulated MB-NPs showed a potent reduction in bothendogenous and overexpressed tau protein levels in human neuroblastomaSHSY-5Y cells expressing endogenous tau and transfected HeLa cellsover-expressing tau protein, respectively. Furthermore, in vitro BBBTranswell study showed significantly higher permeation of MB-NP comparedto the MB solution through the co-culture of rat brain endothelial 4(RBE4) and C6 astrocytoma cells (p<0.05). The MB loaded nanoparticlescould provide a more effective treatment option for AD and many otherrelated disorders.

In an embodiment, a delivery system of coated and uncoated nanoparticleswas developed using a PLGA-based polymer that was repeatedly shown to bebiocompatible and biodegradable. The parameters of synthesized NPs werewithin the suitable range for BBB permeation; specifically, the NPs weremonodispersed, with a slight negative charge, and with the size/diameterwithin 100-150 nm range suitable for intravenous delivery and deliveryto the brain. The coating on the nanoparticle did not have a significantimpact on the nanoparticle size and zeta potential. Based on theimmunoblotting experiments using AD cellular model, the GSH-coated NPswere better in reducing tau levels compared to MB solution. In vitro BBBTranswell permeation study showed an eight-fold higher MB-NP permeationcompared to the MB solution over 24 hours.

As used herein, the term “nanoparticle” refers to any polymeric micelle,lipid micelle, hybrid lipid-polymer micelle, liposome, niosomes,transferosome, liponanoparticle, lipid nanoparticles, nanostructuredlipid nanocarriers (NLC), solid lipid nanoparticles (SLN), hybridlipid-polymer nanoparticles, bicelle, polymerosomes, lamellarstructures, and lipid vesicles, among other delivery systems that can beused suitably to deliver an active pharmaceutical agent, such asmethylene blue.

Examples of polymers used to prepare nanoparticles include, but are notlimited to, lipids or oils, gelatin, sodium alginate, gum arabic,starch, tragacanth, shellac, paraffin wax, poly (lactide-co-glycolide)(PLGA), polylactic acid (PLA), polycaprolactone (PCL), methyl cellulose,pectin, carrageenan, alginates, methyl cellulose, casein, bovine albuminserum, chitosan, hydroxypropyl cellulose, hydroxypropyl methylcellulose, ethyl cellulose, cellulose acetate phthalate, carmellose,polyvinyl alcohol, polystyrene, polyurethane, polyvinylpyrrolidone,polymethacrylate, polyvinyl acetate, polyhydroxyethyl methacrylate,polyvinyl chloride, polyacrylate, polyacrylamide, polyethylene glycol,polyester, polyurea, and polyamide, among other suitable polymers thatmay be used to prepare the nanoparticles.

Examples of lipids that may be used include, but are not limited to,derivatives of glycerophospholipids, glycerolipids, sphingolipids,sterols, fatty acyl amides, prenols, ceramides, cholesterols, lecithin,glyceryl behenate (COMPRITOL), glyceryl palmitostearate (PRECIROL),glycerol monosterol (MONOSTEROL), glycerol disterate, sulfatides,phosphosphingolipids, phosphatidylcholines, phosphatidic acids,phosphatidylethanolamines, phosphatidylglycerols, phosphatidylserines,and phosphor lipids, among other suitable lipids that may be used toprepare nanoparticles.

Examples of oils that may be used include, but are not limited to,safflower oil, sesame oil, corn oil, castor oil, coconut oil, almondoil, cotton seed oil, soybean oil, olive oil, mineral oil, spearmintoil, clove oil, lemon oil, peppermint oil, triacetin, tributryin, ethylbutyrate, ethyl caprylateoleic acid, ethyl oleate, isopropyl myristateand ethyl caprylate, among other suitable oils that may be used toprepare nanoparticles.

The nanoparticles can be prepared using electrostatic interaction,self-assembly, ionotropic gelation, cross-linking, coacervation,homogenization-solvent evaporation, sonication, ultrasound,nanoprecipitation, spray drying, high pressure homogenization, layer bylayer, freeze drying, hot-melt homogenization, film formation,co-solvent evaporation, high pressure instruments such as NANODEBEE, andcoating or solvent emulsion methods, in combination or alone.

The surface of the nanoparticles can be modified with spacer linked to ahydrophilic group, enabling penetration of the BBB. Examples of spacersthat can be used include, but are not limited to, hydrocarbon chainshaving one or more double bonds or triple bonds. More specifically,spacers may be modified or unmodified polyethylene glycols, propyleneglycols, amino acids, peptides, chelators and polaxamers, among otherknown suitable spacers. Examples of chelators include, but are notlimited to, saccharides, urea, DTPA, methyl cellulose, polyvinylpyrrolidone, 1,2-Dioleoylsn-glycero-3-[(N-(5-amino-1-carboxypentyl)imidodiacetic acid) succinyl nickel salt] (DGS-NTA (Ni)) and EDTA, amongother suitable chelators.

If a hydrocarbon chain is present, the hydrocarbon chain may beinterrupted by —O—, —S—, —N(R)—, —N(R)—C(O)—O—, —O—C(O)—N(R)—,—O—C(O)—O—N(R)—, —N(R)—C(O)—N(R)—, —O—C(O)—O—, —P(R)—, —P(O)(R)—, and—C(O)—O(R)—. Each R, independently, may be aliphatic or aromatic. Ifaromatic, the compounds may include two (2) or more membered rings withor without heteroatoms. Examples of R can include, but would not belimited to, hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl,hydroxyl, and haloalkyl. The interrupted hydrocarbon chain may be usedfor conjugation with each polymer or lipid.

As will become clearer as this specification continues, drug releasefrom the nanoparticles can be immediate release or controlled releasefor the drug from the layer of nanoparticles. Particular polymers and/orlipids can be selected for immediate or controlled release of each drugfrom the layer of nanoparticles.

In the following non-limiting study, uncoated and glutathione-coated NPscontaining MB were prepared and characterized for particle size,stability testing, in vitro drug release study, and their tau reducingfunction in cellular models of AD and other tauopathies. Althoughmethylene blue is discussed herein as the pharmaceutical agent, anysuitable pharmaceutical agent is contemplated by the current invention,whether used in tau level reduction or not.

Development of Coated and Uncoated MB-NPs, and Analysis Thereof

Blood-brain barrier (BBB) permeation is one of the key challenges in thefield of pharmacy as most of large molecule pharmaceutics are incapableof crossing BBB [48-50]. Some of the drug delivery systems actually relyon the BBB disruption for permeation and delivery, especially inpresence of solvents such as sodium dodecyl sulfate (SDS), ethanol,dimethylsulfoxide (DMSO), glycerol, and polysorbate-80 (TWEEN 80), whichare often used in drug formulations [50-52]. However, disruption to theBBB may present toxicity as it may allow penetration of bloodcomponents, such as albumin, which are highly toxic, to the brain. Inthe current study, MB-NPs were developed to be used potentially for thetreatment of Alzheimer's disease and other tauopathies. Theglutathione-coated NPs were synthesized for the enhanced brainpermeation due to abundance of GSH transporters present on the BBB[20-22, 31-34](FIGS. 1 and 2). The physical parameters of the NPs werestudied in detail to ascertain suitability of the NPs properties fordelivery across the BBB.

The average sizes/diameters of the NPs were within the 100-150 nm sizerange, which is appropriate for intravenous administration and bloodbrain barrier permeation [30, 53](Table 1). The effect of the drugloading and the GSH coating on the average size of the nanoparticles wasstudied by light scattering, demonstrating decrease in size withMB-loading. The presence of GSH coating did not significantly alter thesize of the NPs, which is important for tight control of the NPscharacteristics to avoid potential unexpected adverse effects that mayarise with variation of the NPs parameters, as the size of thenanoparticles can be an important factor in determining their uptake andtoxicity, thus, narrow distribution of size may be desired [19, 31].

Further, the developed NPs had a slight negative charge as indicated bythe negative zeta potential (Table 1). Presence of a slight negativecharge allows the NPs to disperse in the solution and thus would remainin the blood stream for BBB permeation. The electrostatic repulsionprevents agglomeration that may potentially alter the interactions ofNPs with their environment, which can, in turn, potentially inducetoxicity [32].

The NPs demonstrated the ability to be stable in the PBS solution in thespan of 5 days; the stability of the delivery system can be veryimportant for successful targeting and crossing of the BBB (Table 2). Nosignificant changes in the NPs sizes were detected. However, change inzeta potential was observed, perhaps due to the interactions of thenanoparticles with the salts of the PBS buffer. Unexpectedly, the GSHcoating appears to have contributed to stability of the NPs, as thechange of charge of the GSH-coated NPs was less drastic compared to theuncoated MB-NPs in the span of 5-day stability study (Table 2).GSH-coated nanoparticles demonstrated the least change of zetapotential—a finding consistent with the presence of GSH coating, whichmay limit the interaction of the NPs surface with its environment. Theimplications of maintenance of an intact GSH coat may be effective fortargeted delivery and uptake of MB-NPs in the brain.

The NPs with entrapped drug are postulated to release the drug bydiffusion, by surface erosion or bulk erosion of the polymer, and/or byswelling [54, 55]. There are three main stages of drug release: initialrelease phase, sustained release phase, and final polymer erosionaccelerated phase. During the initial release phase, the drug on thesurface of the NPs is released into the release medium. The sustainedrelease phase follows due to diffusion of the drug out of the NPs, whichis mainly influenced by the molecular weight of the drug and theporosity of the NPs wall. Finally, the accelerated phase occurs as thepolymer erodes in connection with the glass transition temperature ofthe polymer and the interaction of the drug with the polymer. Therefore,the rate of release can be regulated by adjustment of the parametersrelating to the properties of the NPs wall, such as polymer chainlength, flexibility, mobility, swelling, and/or interactions between theactive ingredient and the polymer.

Both coated and uncoated MB-NPs formulations showed no burst drugrelease, which may be important for attaining sustained slow release forthe optimal therapeutic benefit in treatment (FIG. 3). The initialrelease phase of up to 6 hours is most likely due to shedding of the MBfrom the surface of the NPs as the rate of the release is faster thanthat of the control, where the MB is trapped in the mixture of thepolymer. The latter phase (after 8 hours demonstrating slow andsustained release) may be attributed mainly to the diffusion of the MBout of the nanoparticle and partial disintegration of polymer enclosure.The initial higher release for uncoated nanoparticles may be due to thedrug present on the surface of the nanoparticles. This was not seen inthe physical mixture or in the GSH-coated nanoparticles (as the drug onthe surface was coated by the glutathione).

The GSH-coated nanoparticles exhibited a very different release profilecharacterized both by the absence of the initial rapid release phaseseen in uncoated MB-NPs and by the presence of progressive slow release.These findings are consistent with the assumption of formation of theglutathione coating. Specifically, because the MB on the surface of theNPs are displaced by the GSH coating, the initial phase of fast releaseis not observed. Moreover, the release profile shows that GSH decreasesthe release rate, perhaps by partially sealing the NPs and slowing downthe diffusion of MB out of the NPs, which is evidenced by the slowerrelease rate of MB from GSH-coated NPs than both the controlnanoparticles and the uncoated nanoparticles. Overall, GSH-coatednanoparticles demonstrated the necessary controlled and sustainedrelease of MB for the delivery of MB through the BBB, while maintainingtissue saturation levels in the brain.

The effects of NPs on the ability of MB to reduce tau levels were testedin well-characterized cellular models of AD and tauopathies as well [44,45]. Treatment of GSH-coated MB-NPs showed greater reduction in taulevel compared to the MB solution (FIG. 4), demonstrating the capacityof the nanoparticles to effectively allow for delivery of the MB to thecell without compromise in its activity. The more effective reduction intau levels by GSH-coated NPs may be, perhaps, due to the drasticincrease of the reactive surface area as provided by the NPs enclosure,facilitating improved uptake by the cells [30, 40]. Furthermore, a BBBpermeation study by using in vitro Transwell system indicated anapproximately 8-fold higher permeation of 2% glutathione coated MB-NPcompared to the MB solution over 24 hours through the co-culture of RBE4and C6 cells [46, 47].

Overall, the findings of the study provided a valuable tool and atherapeutic option for the treatment of AD and other relatedtauopathies. The findings suggest that newly-prepared MB nanoparticlesare functionally as effective as MB alone.

Materials and Methods

Materials

PLGA-PEG-COOH (RESOMER RGP d 50105, copolymer ratio 50:50, PEG content5%) was obtained from BOEHRINGER INGELHEIM CHEMICALS, Inc., Petersburg,Va., USA. Methylene blue was obtained from Sigma. Glutathione (reduced)was obtained from MP BIOMEDICALS, LLC, Solon, Ohio. NaCl, KCl, Na₂HPO₄,and KH₂PO₄ for phosphate buffered saline (PBS) preparation were obtainedfrom SIGMA-ALDRICH Co., St. Louis, Mo., USA. Acetonitrile, acetone,methanol, trifluoroacetic acid, and triethylamine were HPLC gradepurchased from SIGMA-ALDRICH Co., LLC, USA.

All other chemicals used in the study were of analytical grade and wereused without any further purification unless specified. Anti-Tauantibody and Anti-GAPDH antibody were purchased from SANTACRUZ BIOTECHand MERIDIAN LIFE SCIENCES, respectively. Cell culture reagents and celllysis buffer were purchased from INVITROGEN and FISHER SCIENTIFIC,respectively, as described earlier [41].

Preparation of Glutathione Coated PLGA-PEG NPs

NPs made using PLGA-PEG-COOH polymer were synthesized by ananoprecipitation method described in the art [12, 30]. Briefly, four(4) mg of methylene blue and 100 mg PLGA-PEG-COOH were dissolved in 3 mLof acetone with addition of 100 μL of EtOH to facilitate dissolving ofMB. The solution was then added drop-wise into 6 mL of 1% polyvinylalcohol (PVA) solution, giving a final NPs concentration of 16.67 mg/mL.The optimal ratios of about 0.04 for MB/polymer and about 0.5 foroil/water were maintained for synthesis of the nanoparticles in thisexperiment. Acetone was then removed from the emulsion by evaporationwhile stirring at 40° C. To remove unencapsulated drug and emulsifier.NPs were collected by centrifugation at 18,000×g for 35 min. The pelletwas suspended in PBS pH 7.4 by sonication and used for further analysis.In order to get 2% glutathione coating, twenty (20) mg glutathione wasadded per 1 mL of NPs (16.667 mg/mL) and incubated at room temperaturefor at least 30 minutes before use. The resulting structure can be seenin FIG. 1.

Effects on Particle Size

The particle size and zeta potential of uncoated and glutathione coatedPLGA-PEG NPs were measured by the degree of light scattering usingMICROTRAC FLEX, MICROTRAC, Inc., PA, USA. The polydispersity index wasmeasured using a DYNAMIPRO plate reader, Wyatt Technology, CA, USA. TheNP samples were diluted to fit instrument specifications. In measuringthese parameters, the effect of MB loading into the nanoparticle and theeffect of presence of reduced glutathione coating on mean particle sizeand zeta potential of the NPs were studied. Particle size stability wasmeasured by suspending the NPs in PBS and incubating at room temperature(22° C.) for 5 days.

Determination of Entrapment Efficiency (6)

Entrapment efficiency was determined using a method taught in the art[25, 30]. In brief, standard solutions of methylene blue (MB) inmethanol were measured by a NANODROP spectrophotometer (NANODROPTECHNOLOGIES Inc., DE USA) at a wavelength of 600 nm to obtain thecalibration curve of the drug [42]. The NPs were collected bycentrifugation (18,000 rpms for 35 min) and the supernatant removed. Thepellet of NPs was solubilized in methanol and allowed to dissolveovernight to extract the drug. The sample was again centrifuged toremove any polymer from the solution, and the amount of solubilized drugwas determined by spectroscopy. For the entraption efficiency ofglutathione-coated NPs, the pH of the pellet solubilized in methanol wasadjusted with access of NaOH to facilitate transformation of clearreduced leuko-MB form to blue MB for quantification by thespectrophotometry. From the standard concentrations in methanol(r²=0.99997), a standard curve was determined and the entrapmentefficiency was calculated. All experiments were repeated three times andaverage values were used. The entrapment efficiency (%) was calculatedusing the following equation:

${{Entraption}\mspace{14mu}{efficiency}\mspace{14mu}\%} = {\frac{{MB}_{NP}}{{MB}_{free} - {MB}_{NP}} \times 100}$where MB_(NP) represents the amount of methylene blue trapped in thenanoparticle and is quantified from solubilizing the pellet. MB_(free)corresponds to the amount of free methylene blue in the supernatant.

Transmission Electron Microscopy

Examination of nanoparticles morphology, size and shape, was conductedvia transmission electron microscopy (TEM) using a JEOL 100S TEM (JEOLLtd, Tokyo, Japan). Following particle sizing, the sample was diluted100 times, and ten (10) μL of the NPs solution was carefully placed ontoa copper grid coated in a nitrocellulose membrane.

In Vitro Drug Release

The MB release rate from the NPs was measured in PBS (pH 7.4). MB-loadedNPs were placed into a dialysis bag (MWCO 1,000) and suspended in 80 mLof PBS buffer pH 7.4 at 37° C. and stirred at 60 rpms. Samples of 2 mLwere collected at predetermined time intervals, and the same volume wasreplaced with fresh PBS. The volume of each collected fraction wasreduced to 250 μL, and the samples were then subjected to analysis byhigh performance liquid chromatography (HPLC) in duplicate measurementsto assess the amount of methylene blue released. Aliquots were analyzedon AGILENT INFINITY 60000 (AGILENT TECHNOLOGIES, CA, USA) using reversephase Ascentis C-18 100×4.6 mm column with pore size of 2.7 μm.

A modified method was used for the analysis with mobile phase including80% acetonitrile, 20% water, and 0.1% trifluoroacetic acid, pH 3.0,adjusted with thriethyamine [43]. The flow rate was 0.5 mL/min withinjection volume used of 15 μL.

After obtaining a chromatogram, the peak areas were converted toconcentrations using a standard curve analyzed simultaneously(r²=0.99673). The percent drug release was then calculated for eachsample. Each experiment was repeated three times with technicalduplicates, and the average values were used.

Cell Culture, Treatment and Immunoblotting

Endogenous Tau expressing human neuroblastoma SHSY5Y cells and Helacells stably overexpressing wild-type human tau were maintained inOptiMEM media supplemented with fetal bovine serum and antibioticsolution as described previously [44]. In six-well plates, cells weretreated with control or MB-NPs for 24 hours. Cells were harvested andprocessed for immunoblotting as described previously [45].

In Vitro Blood-Brain Barrier Permeation Assay:

Transwell Permeable Supports (CORNING) with a 0.4 μm pore size werepurchased to investigate the permeability of MB nanoparticles across theblood-brain barrier (BBB) [46, 47] (FIG. 5). Each side was coated with0.1% rat tail collagen I solution diluted from the purchased 100 mgsolution (Collagen I, rat tail, 100 mg, BD BIOSCIENCES), left under thehood to adhere to the support for 1 hour, and washed with 200 ML of1×PBS after aspiration of the remaining collagen solution. The mediaused was a 1:1 solution of Ham's F10:MEM media (CELLGRO) supplementedwith 10% fetal bovine serum (FBS) (INVITROGEN), 1% penicillin andstreptomycin (CELLGRO), 0.4 mL of 1 ng/mL human recombinant dilutedbasic fibroblast growth factor (bFGF) (BD Biosciences), 5 mL of HEPESbuffer (FISHER), and 2 mL of a 200 mM stock of L-glutamine (MEDIATECH).1.5 mL of media was added surrounding the support, and 0.5 mL was addedinside the support. This was left to incubate in the incubator (37° C.and 5% CO₂) for 24 hours.

After 24 hours, the media was aspirated from the plates. The co-culturewas prepared by first seeding 5×10⁴ C6 rat astrocytic cells (ATCC,CCL-107) to the bottom of the support, which remains under the hood for1 hour, and is subsequently transferred back to the incubator for 2hours. Next, the remainder media was aspirated, 1.5 mL of media wasadded to the plate, and the support was reverted back to fit into thewell; 0.5 mL of media was added back to the inside of the support. After48 hours of incubation, the media was aspirated from the inside of thesupport and 5×10⁵ rat brain endothelial cells (RBE4 cells, gifted by Dr.Aschner of Vanderbilt University Medical Center) were seeded to theinside. 0.5 mL of media was made up after the RBE4 were seeded to theinside of the insert and the plate was placed back in the incubator for24 hours to allow the cells to adhere (FIG. 5).

Subsequently, the media was aspirated and replaced with equal amounts of1% FBS experimental media. The co-culture was treated by adding thefollowing to the 0.5 mL of media inside the support: 10 μM of MB drug insolution; uncoated MB-loaded NPs; and 2% glutathione-coated, MB-loadedNPs. Four (4) samples of 100 μL media were taken out from under thesupport at approximately 0, 3, 6, and 24 hours and fresh experimentalmedia was used to replace the extracted volume. The samples werequantified by UV spectrometry in the SYNERGY H4 plate reader (BIOTEKINSTRUMENTS Inc.) at a wavelength of 600 nm. Data was quantified intriplicates and averaged (n=3).

Statistical Analysis

The data were expressed as the mean of at least three experiments±standard error.

Statistical comparison between the controls and methylene bluenanoemulsion was performed with paired student t-test to assess thesignificance of differences. The in vitro release profiles of the NPsand the control were compared by two-way ANOVA analysis usingstatistical analysis software SAS (SAS INSTITUTE Inc., NC, USA). A fivepercent (5%) significance level (p-value ≤0.05) was utilized for allstatistical analyses.

Results

Physical Parameters of the Nanoparticles

In order to understand the effect of MB loading and GSH coating on theNPs parameters, the physical characteristics of the NPs, such as size,polydispersity index, were evaluated (see Table 1). MB-NPs and coatedMB-NPs were compared to the blank NPs containing no drug, which wasutilized as a control. Investigation of the sizes of MB-loaded NPs andblank NPs revealed that the average sizes/diameters of each as 150±10 nmand 147.7±4.84 nm, respectively. The average size is the result ofquantifying triplicates and obtaining the standard error. The developedNPs demonstrated reduction in size with drug loading, which may be dueto more formation of a more compact nanoparticle resulting from theinteractions of the polymer with MB. Addition of the GSH coating did notappear to have an effect on the NPs size, where the mean sizes ofuncoated NPs (136.5±4.4 nm) and coated NPs (137.8±6.3 nm) were virtuallyidentical. Further, as compared to the control, NPs with 2% glutathione(GSH) coating did not demonstrate a significant change of the mean size(137.8±6.3 nm), perhaps due to more variations in the size attributed tothe coating layer [25].

TABLE 1 Nanoparticie characterization. Nanoparticles loaded withMethylene blue showed significant decrease in the NPs size (p-value =0.008). Glutathione coating did not significantly alter the nanoparticiesize. Zeta potential in MB-NPs did not significantly change as comparedto blank NPs. All values shown as a mean of triplicates with ± SE. Theacronyms are as follows: nanoparticle (NPs), Methylene blue (MB),glutathione reduced (GSH), polydispersity index (PDI), Entraptionefficiency (EE). Zeta potential Nanopartieles Size (nm) PDI mV EE %(NPs) Mean ± SE Mean ± SE Mean ± SE Mean ± SE NPs 147.7 ± 4.84   0.014 ±0.0003 −19.21 ± 1.15 NA MB-NPs 150 ± 10  0.052 ± 0.013  −7.4 ± 1.3925.07 ± 3.19 2% GSH-coated 137.8 ± 6.3  0.052 ± 0.013  −1.84 ± 3.6719.13 ± 0.05 MB-NPs

Zeta potential describes the surface property of the NPs and is animportant factor contributing to the biointeractions of the NPs with itsenvironment [32, 33]. In the current study, NPs, MB-NPs, and coatedMB-NPs exhibited negative zeta potential, as follows: −19.21.15 mV,−7.4±11.30 mV, and −1.84±3.67 mV, respectively (Table 1).

In order to ascertain that the batch includes monodisperse NPs ofconsistent and uniform size, the polydispersity index (PDI) was obtained(Table 1). All NPs formulations followed normal/Gaussian distribution ofparticle size with PDI value below one (1), confirming narrowdistribution of the size of the NPs.

Due to its hydrophilic nature, MB readily dissolves in water phase ofthe nanoprecipitation approach to synthesis of NPs, thus presenting achallenge to encapsulation by the polymer. However, with the currentnanoprecipitation method, a 25.07±3.19% encapsulation of the drug wasachieved (see Table 1). The addition of GSH coating may be seen to lowerthe amount of measured encapsulated drug as the coating replaces the MBinevitably adsorbed at the surface of the NPs.

Effective API delivery requires system stability in physiologicalsolutions. To assess the nanoparticle stability, both coated anduncoated NPs were kept in PBS for at least 5 days, and changes in themean size and zeta potential were measured (Table 2). No significantchanges in the mean particle sizes of MB-NPs and coated MB-NPs weredetected. However, GSH-coated NPs decreased in mean size by about 12%,and MB-NPs decreased in mean size by about 2%. The larger difference inmean size after 5 days in GSH-coated NPs confirms the presence of theGSH coat on the NPs and could be attributed to gradual shedding of GSH.Because the changes in mean size were not significant (p-value≤0.05)(i.e., GSH coating is not lost even after 5 days), however, it would beexpected that the GSH coating would remain mostly intact in aphysiological system.

TABLE 2 Stability of the nanoparticles. Methylene blue nanoparticlesexhibited stability over the period of 5 days. The changes in size forall NPs were not significant with p-value ≥ 0.05. Glutathione-coated NPsshowed the most of size change due to partial shedding of the GSH coat.All values shown as a mean of triplicates with ± SE. Nanoparticle (NPs),Methylene blue (MB), glutathione reduced (GSH), polydispersity index(PDI). Day 1 After 5 days NPs size NPs zeta NPs size NPs zeta Change inNanoparticles (nm) potential (mV) (nm) potential (mV) mean size, %MB-NPs 136.5 ± 4.4  −7.4 ± 11.39  135 ± 21.45 23.2 ± 12.02  2%GSH-coated 137.8 ± 6.35 −1.83 ± 3.67  121.4 ± 5.24  2.07 ± 7.87  12%MB-NPs

NPs Morphology by TEM

Morphology of the MB-NPs was confirmed by transmission electronmicroscopy (TEM) (FIG. 2). TEM imaging confirmed the nanoparticle sizebelow 200 nm. NPs appeared to have spherical morphology with smoothouter surface and to have consistent size throughout the sample. The TEMallowed for visualization of loading of MB inside of the NPs.Additionally, the GSH coating is visible in causing the nanoparticle toreact differently to the beam, resulting in sharper imaging of thenanoparticle edges and of the protrusions on the nanoparticle surface.

In Vitro Release

In this study, the release profile of coated and uncoated MB-NPs wasstudied in vitro and compared to a control, a physical mixture of thepolymer and MB (FIG. 2). Both coated and uncoated NPs demonstrated noburst release. However, the release profile of the uncoatednanoparticles was significantly faster as compared to the control(p-value<0.001) with approximately 60% of MB released within 10 hours;after approximately 10 hours, the release of MB appears to proceed at aslower rate. Through the duration of the study, the control released MBat approximately constant rate by diffusion of MB out of the mixture.GSH-coated NPs exhibited a slower release as compared to the control anduncoated nanoparticles. For about 8 hours of release, the coated NPsdemonstrated significantly slower release rate as compared to theuncoated NPs (p-value<0.001). After 24 hours, the release of MB fromcoated NPs appeared to be comparable to that of the uncoatednanoparticles. Thus, the release of the drug from the GSH-coated NPs canbe characterized as a slow and sustained release for up to 120 hours.

Effect of Nanoparticles on Tau Levels in Cultured Cells

To test the functionality of newly generated NPs, two different cellculture-based AD models were utilized. Endogenously tau expressing SHSHYneuronal cells and stably tau overexpressing HeLa cells were treatedwith 5 μM and 10 μM of MB-NPs, coated MB-NPs, control NPs, vehicle,and/or drug MB-solution for 24 hours. Cells were harvested, and sampleswere analyzed for tau levels by immunoblotting (FIG. 4). The GSH-coatedMB-NPs formulation showed greater reduction in tau level compared to MBsolution (FIG. 4) at 5 μm and 10 μm concentrations in both endogenoustau and over-expressing tau cell lines. The tau reduction level wasalmost similar for coated and uncoated NPs in both cell lines.

In Vitro Blood-Brain Barrier Permeation Assay

FIGS. 6 and 7 illustrate the data from the in vitro BBB permeation ofthe MB drug solution and the glutathione-coated nanoparticles. Ten (10)μM of the 2% glutathione-coated nanoparticles showed the highest levelof permeation across the simulated BBB. There was a linear increase inthe permeation of glutathione-coated nanoparticles permeating the BBBover 24 hours (FIG. 6). The glutathione-coated nanoparticles were seento be more effective at permeating the BBB model than the free MB drugsolution (FIG. 7); data has statistical significance with p<0.05. Thedata of the permeation of the free MB drug solution was decreased by 75%to mimic the approximately 25% entrapment efficiency exhibited by theMB-loaded nanoparticles, as described previously. While the permeationof glutathione-coated MB nanoparticles increased linearly over the24-hour period, the permeation of the free MB drug solution remainedrelatively constant over the same period.

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All referenced publications are incorporated herein by reference intheir entirety. Furthermore, where a definition or use of a term in areference, which is incorporated by reference herein, is inconsistent orcontrary to the definition of that term provided herein, the definitionof that term provided herein applies and the definition of that term inthe reference does not apply.

GLOSSARY OF CLAIM TERMS

Controlled and sustained release: This term is used herein to refer torelease or delivery of pharmaceutical agent in response to a stimulus ortime. As seen herein, a controlled and sustained release ofpharmaceutical agent (e.g., methylene blue) was achieved through theglutathione coating that breaks down over time to release thepharmaceutical agent contained within the layer of nanoparticles.

Extends outwardly: This term is used herein to refer to projectingexternally from the surface of an object. Thus, a spacer and connectedhydrophilic group—that extends outwardly from a layer of nanoparticlesthat substantially encloses a pharmaceutical agent-projects externallyaway from the interior of that enclosure, such that the hydrophilicgroup can interact with the external environment prior to thenanoparticles.

Hydrophobic, protective coating: This term is used herein to refer to acovering around the outer surface of a layer of nanoparticles, where thecovering is capable of traversing a hydrophobic environment, such as theBBB.

Substantially encloses: This term is used herein to refer to surroundinga majority, or nearly all, of something. For example, a layer ofnanoparticles can substantially enclose a pharmaceutical agent, such asmethylene blue, by surrounding a majority of the pharmaceutical agent.In some situations, as discussed previously, methylene blue has beenseen to diffuse through the BBB prior to breakdown of the polymer-basedlayer of nanoparticles.

Targeted region: This term is used herein to refer to an area within asubject's body in need of delivery of a pharmaceutical agent (e.g.,methylene blue). An example of a targeted region is the brain of asubject.

The advantages set forth above, and those made apparent from theforegoing description, are efficiently attained. Since certain changesmay be made in the above construction without departing from the scopeof the invention, it is intended that all matters contained in theforegoing description or shown in the accompanying drawings shall beinterpreted as illustrative and not in a limiting sense.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention that, as amatter of language, might be said to fall therebetween.

What is claimed is:
 1. A nanoparticle drug composition comprising: atleast one nanoparticle comprising a poly(lactic-co-glycolic acid)polymer conjugated to polyethylene glycol (PEG) linked to a hydrophilicgroup wherein the nanoparticle is coated with glutathione; and ahydrophilic pharmaceutically active agent encapsulated within the atleast one nanoparticle at an entrapment efficiency of about 19% whereinthe hydrophilic pharmaceutically active agent is methylene blue; whereinthe nanoparticle drug composition is generated by a process consistingessentially of: dissolving or diluting the poly(lactic-co-glycolic acid)polymer conjugated to the polyethylene glycol (PEG) linked to thehydrophilic group and a predetermined quantity of the hydrophilicpharmaceutically active agent in an amount of acetone and an amount ofethanol to form a solution; adding the solution to a polyvinyl alcohol(PVA) solution to form an emulsion; evaporating the acetone from theemulsion wherein upon evaporation of the acetone, at least onenanoparticle is generated; centrifuging the emulsion to removeunencapsulated hydrophilic pharmaceutically active agent and the PVA;and coating the at least one nanoparticle with the glutathione.
 2. Thenanoparticle drug composition of claim 1, wherein the hydrophilic groupis a carboxyl group.
 3. The nanoparticle drug composition of claim 1,wherein the at least one glutathione-coated nanoparticle has a diameterof between about 100 nm to about 150 nm.
 4. The nanoparticle drugcomposition of claim 1, wherein the ratio of the hydrophilicpharmaceutically active agent to the poly(lactic-co-glycolic acid)polymer is about 0.04.
 5. A method of reducing tau levels in a brain ofa patient suffering from Alzheimer's disease or other related taupathiescharacterized by increased tau levels comprising: administering atherapeutically effective amount of a nanoparticle drug compositionaccording to claim 1 wherein the hydrophilic pharmaceutical agent ismethylene blue.
 6. The method of claim 5, wherein the hydrophilic groupis a carboxyl group.
 7. The method of claim 5, wherein the disease isAlzheimer's disease.
 8. The method of claim 5, wherein the nanoparticleshave a diameter of between about 100 nm to about 150 nm.